Prosaposin deficiency and saposin B deficiency (activator‐deficient metachromatic leukodystrophy): Report on two patients detected by analysis of urinary sphingolipids and carrying novel PSAP gene mutations

Abstract

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65–70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A–D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated. © 2009 Wiley-Liss, Inc.

Tandem Mass Spectrometry of Sphingolipids: Application in Metabolic Studies and Diagnosis of Inherited Disorders of Sphingolipid Metabolism

“…Extracts were prepared as previously published (Kuchar, et al, 2009). First, 150 µl of sonicated urine was extracted with 700 µl of chloroform:methanol (2:1, v:v) containing internal standards in a polypropylene Eppendorf tube.…”

“…Corresponding internal standards were added to the urinary samples during the extraction process (Kuchar, et al, 2009). The same volume of internal standards as was used for the urine samples was added to the appropriate aliquots of purified lipid extracts: 5 µg of cellular protein or 150 µg of tissue protein.…”

“…The resolution was generally set to unit (±1 m/z), but in some cases, we used a high resolution setting for the first quadrupole, as using such a setting can improve mass spectrometer sensitivity. Procedure of our quantitative analysis was described in a previous study (Kuchar, et al, 2009). Problems associated with the matrix effect were addressed using internal standards, whereas the calibration of the method was based on an external standard.…”

“…8B). It is also possible to use the ratio of the analyzed compound to sphingomyelin (Berna, et al, 1999;Kuchar, et al, 2009) or phosphatidylcholine (Fuller, et al, 2005;Whitfield, et al, 2001), which are membrane-bound lipids that can be measured simultaneously in the same sample. …”

“…These disorders are characterized by the massive excretion of specific sphingolipids, e.g., Gb3Cer in Fabry disease ( -galactosidase A deficiency due to mutations of the GLA gene); multiple hydrophobic sphingolipids in complex sphingolipidoses, in which the defect is caused by mutations in the prosaposin gene (sphingolipids with a saccharide chain that is shorter than four monosaccharide units and ceramides are not degraded in prosaposin deficiency and Gb3Cer and sulfatides in saposin B deficiency due to defective activator proteins); sulfatides in metachromatic leukodystrophy (arylsulfatase A deficiency due to mutations of the ARSA gene) (Fuller, et al, 2005;Kuchar, et al, 2009;Whitfield, et al, 2001). We developed a method of tandem mass spectrometry quantification of urinary sphingolipids that can be used in pre-diagnostic screening for the lysosomal disorders mentioned above (Kuchar, et al, 2009). Our data are presented in Analyzing non-degraded metabolites can be very helpful in the pre-diagnosis of sphingolipid activator deficiencies in which routine enzymology fails to indicate deficient enzyme activity due to the detergents commonly used in the assays.…”